Fluorescence Characterization of Internally Labeled DNA with Fluorescently Tagged Nucleotides

Ahmed Heikal¹, Jonas Korlach¹, Dan W. Baird², Gregory R. Hoffmann², Kyle Gee³, Richard A. Cerione², and Watt W. Webb¹

Dense internal labeling of DNA with fluorescent markers has numerous applications that include PCR, karyotyping, comparative genomic hybridization, and DNA sequencing. A fundamental understanding of the molecular spectroscopy, dynamics, and structures of these labeled biopolymers is essential for advancing those applications. Here, we replace the native deoxyribonucleotide triphosphate (dNTP) in a DNA molecule with fluorescently tagged dNTPs using polymerase-mediated DNA synthesis. The effects of linker length, base type, and DNA environment on the label-fluorescence properties will be discussed using time-resolved fluorescence and anisotropy. Comparative studies on YOYO-1 intercalated with DNA further demonstrate that local mobility of the DNA can be monitored with fluorescently labeled DNA. Finally, GTP analogs in which the labels are coupled to the -phosphate are investigated as function of binding to different G proteins that play fundamental role in signal transduction and cancer research. Financial Support (Grant Number): NCRR-NIH (P41-RR04224) and NSF (P412 RR04224).

¹ School of Applied and Engineering Physics
² Molecular Medicine, Cornell University, Ithaca, NY
³ Molecular Probes, Eugene, OR